apoptosis upon over-expression with pcDNA6-cDNA - how prove sequence specificit
Posted 22 June 2011 - 01:26 PM
I am trying to establish a cell line stably over-expressing my gene of interest. Transfecting the cDNA in a pcDNA6-plasmid lead to apoptosis of the cells with a 3 day delay compared to untransfected controls. Is there any good way to demonstrate clearly that this is sequence specific and definitely not related to transfection efficiency? Or a first step before I'd start cloning my cDNA into other (i.e. inducible) systems or similiar?
Any help would be really appreciated!!
Posted 22 June 2011 - 06:08 PM
Posted 22 June 2011 - 09:07 PM
Did you do an "empty" vector control (i.e. pcDNA6 with no insert) and a non-target vector control (i.e. pcDNA6 with another cDNA or a scrambled version of your cDNA)?
Yes, I had the empty vector, which was proliferating happily under selection but no scrambled cDNA. How could I make a scrambled version of my cDNA?
Thanks so much for any help!
Posted 23 June 2011 - 05:37 PM
You should also check expression of your gene in the cells by western and/or RT-PCR.
Posted 25 June 2011 - 10:58 AM